The polyamines spermidine and spermine, as well as its precursor, putrescine, are required for numerous cellular functions in mammalian cells, including post-translational modification of eukaryotic initiation factor eIF-5A, ion channel gating, and at several steps of nucleic acid and protein synthesis. Polyamines are synthesized by most cell types, and catalysis of putrescine biosynthesis via ornithine decarboxylase1 (ODC) is a major rate-limiting step in the polyamine biosynthetic pathway. Polyamines can also be utilized from extracellular sources via one or several membrane carriers (Seiler et al. (1990) Int. J. Biochem. 22, 211-218). A number of polyamine carriers of various substrate specificity have been characterized from bacterial species, and a vacuolar polyamine transporter has been identified in the yeast Saccharomyces cerevisiae. However, no molecular identification for a plasma membrane transporter specific for polyamines has yet been reported in eukaryotes.
High-affinity mammalian polyamine transport activity is membrane potential-dependent and Na+ gradient-independent, and requires divalent cations (e.g. Ca2+, Mg2+, Mn2+) for its activity (Poulin et al. (1998) Biochem. J. 330, 1283-1291). Polyamine uptake is regulated by intracellular polyamines through several feedback mechanisms (Seiler et al. (1990) Int. J. Biochem. 22, 211-218), including rapid down-regulation by ODC antizyme via translational frameshifting (Matsufuji et al. (1996) EMBO J. 15, 1360-1370) and up-regulation of its Vmax upon chronic polyamine depletion (Seiler et al. (1990) Int. J. Biochem. 22, 211-218; Lessard et al. (1995) J. Biol. Chem. 270, 1685-1694) by agents like α-difluoromethylornithine (DFMO), a suicide substrate of ODC (Lessard et al. (1995) J. Biol. Chem. 270, 1685-1694). DFMO is currently commercialized under the trade name of Eflornithine™. Moreover, enhanced polyamine transport is associated with rapid cell proliferation and transformation (Seiler et al. (1990) Int. J. Biochem. 22, 211-218).
The absolute polyamine requirement for tumor progression has been the target of promising therapeutic approaches such as the vectorization of cytotoxic polyamine analogs such as N1,N11-diethylnorspermine through the polyamine transport system, or polyamine depletion using DFMO (Marton et al. (1995) Ann. Rev. Pharmacol. Toxicol. 35, 55-91). The latter approach is currently considered as a potentially effective treatment for chemoprevention of various cancers in human (Gerner et al. (2004) Nat Rev Cancer. 4:781-792). In addition to promoting cytostasis, polyamine depletion through the use of DFMO inhibits angiogenesis and metastasis (Jasnis et al. (1994) Cancer Lett. 79, 39-43). Although most tumor cell types enter growth arrest upon treatment with DFMO, the in vivo therapeutic efficacy of DFMO has been limited to isolated cases (Jasnis et al. (1994) Cancer Lett. 79, 39-43; Marton et al. (1995) Ann. Rev. Pharmacol. Toxicol. 35, 55-91). There is substantial evidence that the antitumor action of DFMO is severely impaired by the high-affinity capture of plasma polyamines by tumor cells. For instance, tumors formed by polyamine transport-deficient cells are much more sensitive to DFMO in mice than in the case of the parental strain (Persson et al. (1988) Cancer Res. 48, 4807-4811). Moreover, polyamine deprivation by decontamination of the gastrointestinal tract or by feeding a polyamine-poor diet can markedly decreases tumorigenesis and enhance DFMO-induced inhibition tumor progression in vivo (Seiler et al. (1990) Int. J. Biochem. 22, 211-218). Concentrations of polyamines similar to those found in human plasma (0.1-1 μM) are in fact sufficient to completely antagonize the effect of DFMO in human breast cancer cells (U.S. Pat. No. 6,083,496).
Limitation of DFMO action by the high polyamine transport activity found in tumor cells could in principle be alleviated by the use of drugs interfering with polyamine uptake. Such drugs should be endowed with high affinity toward the polyamine carrier and low cytotoxicity, and be poorly cell-permeant (U.S. Pat. No. 6,083,496) in order to preserve the therapeutic benefits of DFMO-induced polyamine depletion (Marton et al. (1995) Ann. Rev. Pharmacol. Toxicol. 35, 55-91). Only few attempts have previously been made to design specific inhibitors of polyamine transport. Linear polypyridinium compounds designed as paraquat surrogates are highly potent inhibitors of putrescine transport (Minchin et al. (1989) Biochem. J. 262, 391-395) but their ability to compete for spermidine or spermine uptake has not been reported. More recently, a high Mr (≈25,000) spermine polymer has been shown to be a high-affinity competitor of diamine and polyamine transport (Aziz et al. (1996) J. Pharmacol. Exper. Ther. 278, 185-192), but its high cytotoxicity is probably the main factor involved in its antitumor action.
Applicants have previously showed that 2,2′-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), obtained through dimerization of two N-(2-mercaptoethyl) spermine 5-carboxamide moieties through a disulfide bridge, leads to a potent cell-impermeant polyamine transport antagonist with a much lower Ki than the parent monomer (U.S. Pat. No. 6,083,496). These results were encouraging but some improvement could be interesting in this particular field.